Cancer is thought to arise from a multi-step process that typically involves multiple genetic mutations leading to uncontrolled cell growth. Many cancers are curable if detected early in their development. For example, colorectal cancers typically originate in the colonic epithelium, and are not extensively vascularized (and therefore not invasive) during early stages of development. The transition to a highly-vascularized, invasive and ultimately metastatic cancer commonly takes ten years or longer. If the presence of cancer is detected prior to extensive vascularization, surgical removal typically is an effective cure. However, colorectal cancer is often detected only upon manifestation of clinical symptoms, such as pain and bloody stool. Generally, such symptoms are present only when the disease is well established, and often after metastasis has occurred. Similarly, with the exception of the Pap smear for detection of pre-malignant cervical lesions, diagnostic screening methods for other types of cancer are best at detecting established disease.
Most diagnostic assays for cancer are invasive, or at least uncomfortable. Invasive procedures range from performing a tissue biopsy to surgery. Cancer screening procedures frequently result in significant patient discomfort. For example, magnetic resonance imaging requires confinement of the patient, and colonoscopy requires sedation. The discomfort associated with typical invasive screening methods reduces patient compliance with routine screening procedures.
Moreover, screening for the early detection of cancer (i.e., prior to the onset of symptoms and/or metastasis) often results in an intolerable level of false positive and false negative results. The likelihood of a false positive test result is a function of the specificity of the test. The specificity of a test (expressed as a percentage) is the likelihood that any individual who is negative for disease tests negative for that disease. On the other hand, false negative results are a property of the sensitivity of the test. Sensitivity (also expressed as a percentage) provides the likelihood that a test for a specific disease will identify as positive an individual having that disease. Thus, 5% of the individuals determined to have a disease using an assay with a specificity of 95% will actually not have the disease. Similarly, an assay with a sensitivity of 95% will incorrectly identify a diseased individual as disease-free 5% of the time.
The problems of sensitivity and specificity are exaggerated in assays for the early detection of cancer because patient samples on which such early detection is performed typically contain relatively small amounts of cancerous cellular material in relation to non-cancerous cellular material. In many cases, patient samples are a heterogeneous mixture of large amounts of normal cells and small amounts of cancerous cells. A good example of such a heterogeneous sample is stool. The typical stool sample contains cells and cellular debris sloughed from the colonic epithelium, by-products of digestion, and bacteria. In its early stages, colorectal cancer is thought to affect only about 1% of colonic epithelial cells. Any attempt to detect nucleic acids from the 1% of affected cells in the heterogeneous background of the stool sample might give rise to very low sensitivities. Attempts to identify the presence of the indicia of cancer in other heterogeneous samples, such as sputum, pus, urine, nipple aspirate, etc., presents similar problems.
Recently, a number of genetic mutations have been associated with cancer. For example, alterations in the p53 gene, the Kras oncogene, and the apc tumor suppressor gene are thought to be participants in the multi-step pathway leading to cancer. It has been suggested that mutations in those genes might be a basis for molecular screening assays for the early stages of certain types of cancer. See e.g., Sidransky, et al., Science, 256: 102-105 (1992). Attempts have been made to identify and use nucleic acid markers that are indicative of cancer. However, even when such markers are found, using them to screen patient samples, especially heterogeneous samples, has proven unsuccessful either due to an inability to obtain sufficient sample material, or due to the low sensitivity that results from measuring only a single marker. For example, simply obtaining adequate human DNA from one type of heterogeneous sample (stool) has proven difficult. See Villa, et al., Gastroenterol., 110: 1346-1353 (1996) (reporting that only 44.7% of all stool specimens, and only 32.6% of stools from healthy individuals produced sufficient DNA for mutation analysis). Other reports in which adequate DNA has been obtained have reported low sensitivity in identifying a patient's disease status based upon a single cancer-associated mutation. See Eguchi, et al., Cancer, 77: 1707-1710 (1996) (using a p53 mutation as a marker for cancer).
Accordingly, there is a need in the art for high-sensitivity, high-specificity assays for the detection of molecular indicia of cancer, especially in heterogeneous samples.